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HCV NS5A inhibitors are the backbone of directly acting antiviral treatments against the hepatitis C virus (HCV). While these therapies are generally highly curative, they are less effective in some specific HCV patient populations. In the search for broader-acting HCV NS5A inhibitors that address these needs, we explored conformational restrictions imposed by the [7,5]-azabicyclic lactam moiety incorporated into daclatasvir (1) and related HCV NS5A inhibitors. Unexpectedly, compound 5 was identified as a potent HCV genotype 1a and 1b inhibitor. Molecular modeling of 5 bound to HCV genotype 1a suggested that the use of the conformationally restricted lactam moiety might have resulted in reorientation of its N-terminal carbamate to expose a new interaction with the NS5A pocket located between amino acids P97 and Y93, which was not easily accessible to 1. The results also suggest new chemistry directions that exploit the interactions with the P97–Y93 site toward new and potentially improved HCV NS5A inhibitors.

Background For treatment of genotype 1a (GT1a) infection with elbasvir/grazoprevir, the German guidelines recommend a differentiated approach depending on baseline viral load (BVL). For low BVL ≤800 000 IU/mL, treatment with 12 weeks elbasvir/grazoprevir should be considered, whereas for high BVL >800 000 IU/mL, this regimen is only recommended in nonstructural protein 5A (NS5A) resistance-associated substitutions (RAS) absence. With present NS5A RAS or when RAS-testing is not available, 16 weeks elbasvir/grazoprevir + ribavirin is preferred. Here, we investigated the adherence to these recommendations and the effectiveness of elbasvir/grazoprevir in a large German Hepatitis C-Registry GT1a cohort. Methods From September 2016 until July 2018, 195 GT1a-infected patients were treated with elbasvir/grazoprevir ± ribavirin for 12-16 weeks. The primary outcome was per protocol SVR12 or SVR24. Results Mean age was 50 years, 89% were male, 19% had cirrhosis, 72% were treatment-naive. Forty-five percent had low BVL ≤800 000 IU/mL, 55% high BVL >800 000 IU/mL, of whom 49 vs. 42% were baseline RAS-tested. Four patients with high (7.7%) and two with low BVL (5%) had NS5A RAS of whom 50% received elbasvir/grazoprevir+ribavirin, respectively. Ninety-four percent of patients with low and 65% with high BVL received elbasvir/grazoprevir without ribavirin. Thirty-five percent of patients with high BVL received ribavirin, mostly without prior RAS-testing. Per protocol sustained virologic response (SVR) by low vs. high BVL was 98.8 and 95.1%. All patients with NS5A RAS achieved SVR. Conclusions In German, real-world most patients received elbasvir/grazoprevir without ribavirin. Ribavirin was mainly added in GT1a patients >800 000 IU/mL, who were not NS5A RAS tested. SVR rates were consistently high and comparable to clinical trial results.

Patients infected with hepatitis C virus (HCV) treated with interferon‐free direct‐acting antivirals may still require ribavirin. However, ribavirin is associated with adverse events that can limit its use. This open‐label, multicentre, Phase 3 study evaluated the safety and efficacy of ombitasvir/paritaprevir/ritonavir + dasabuvir (OBV/PTV/r + DSV) with low‐dose ribavirin for 12 weeks in genotype 1a‐infected patients without cirrhosis. The primary efficacy endpoint was sustained virologic response at post‐treatment Week 12 (SVR12). The primary safety endpoint was haemoglobin

Hepatitis C virus (HCV) infection can lead to chronic infection resulting in severe liver diseases, including cirrhosis and hepatocellular carcinoma (HCC). Chronic hepatitis C (CHC) is characterized by ongoing inflammation and generation of reactive oxygen species (ROS) such as superoxide (O2.-) and hydrogen peroxide (H2O2). NADPH oxidase (Nox) enzymes produce ROS as their primary function and have been associated with oxidative stress in CHC. However, the precise role that Nox enzymes play in the pathogenesis of HCC is still unclear. We show that the structural core protein of genotype 1a HCV is sufficient to elevate Nox1 and 4 mRNA, protein expression, and enzyme activity and H2O2 levels in vitro. Human hepatocytes constitutively expressing core exhibited elevated Nox4 in the nucleus and increased DNA damage markers. Furthermore, the novel Nox-specific inhibitor VAS-2870 was capable of decreasing the core-associated increase in Nox enzyme activity. Altogether, these results highlight the importance of developing therapeutic agents targeting Nox enzymes for use as adjunct treatment of CHC to prevent carcinogenesis.

[Abstract] The presence of resistance‐associated substitutions (RASs) at NS5A region might compromise the efficacy of Direct Acting Antiviral agents (DAAs). HCV resistance at NS5A region is mainly focused on patients with hepatitis C virus (HCV) genotypes 1a (G1a) and 3 (G3) with other factors of poor treatment response (ie cirrhosis, prior treatment‐exposure, or HCV‐RNA >800 000 IU/mL). Herein, we evaluated in a cohort of HCV G1a and G3 infected patients the prevalence of RASs at domain I NS5A using population‐based sequencing and the impact of RASs on the optimization of current therapeutic strategies. The RASs considered as clinically relevant were: M28A/G/T, Q30D/E/H/G/K/L/R, L31M/V/F, H58D, and Y93C/H/N/S for G1a and Y93H for G3. A total of 232 patients naïve to NS5A inhibitors were included (166 G1a, 66 G3). The overall prevalence of NS5A RASs for G1a and G3 patients was low (5.5%) or null, respectively. A high proportion of patients harbored, at least, one factor of poor response (78.9% for G1a, and 75.8% for G3). Overall, the rates of patients harboring NS5A RASs in combination with any of the other factors were low and the vast majority of patients (G1a> 94% and G3 100%) could be treated with standard treatments of 12 weeks without ribavirin. In conclusion, testing NS5A RASs in specific HCV‐infected populations (ie G1a & G3, cirrhosis, prior treatment experienced, HCV‐RNA >800 000 IU/mL) might be useful to optimize current NS5A‐based therapies avoiding ribavirin‐related toxicities, and shortening treatment duration in the majority of patients. Instituto de Salud Carlos III; CPII14/00014 Instituto de Salud Carlos III; PI10/02166 Instituto de Salud Carlos III; FI14/00557 Instituto de Salud Carlos III; PI13/02266 Instituto de Salud Carlos III; CM15/00233 Instituto de Salud Carlos III; PI15/00713 Instituto de Salud Carlos III; PI16/02159 Xunta de Galicia; IN606A-2016/023 Comisión Interministerial de Ciencia y Tecnología (España); RD12/0017/006

High hepatitis C virus (HCV) genetic diversity impacts infectivity/pathogenicity, influencing chronic liver disease progression associated with fibrosis degrees and hepatocellular carcinoma. HCV core protein is crucial in cell-growth regulation and host-gene expression. Liver fibrosis is accelerated by unknown mechanisms in human immunodeficiency virus-1- (HIV-1-) coinfected individuals. We aimed to study whether well-defined HCV-1a core polymorphisms and genetic heterogeneity are related to fibrosis in a highly homogeneous group of interferon-treated HIV-HCV-coinfected patients. Genetic heterogeneity was weighed by Faith’s phylogenetic diversity (PD), which has been little studied in HCV. Eighteen HCV/HIV-coinfected patients presenting different liver fibrosis stages before anti-HCV treatment-initiation were recruited. Sampling at baseline and during and after treatment was performed up to 72 weeks. At inter/intrahost level, HCV-1a populations were studied using molecular cloning and Sanger sequencing. Over 400 complete HCV-1a core sequences encompassing 573 positions of C were obtained. Amino acid substitutions found previously at positions 70 and 91 of HCV-1b core region were not observed. However, HCV genetic heterogeneity was higher in mild than in severe fibrosis cases. These results suggest a potential utility of PD as a virus-related factor associated with chronic hepatitis C progression. These observations should be reassessed in larger cohorts to corroborate our findings and assess other potential covariates. Fil: Parra, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; Argentina Fil: Laufer, Natalia Lorna. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: Manrique, Julieta Marina. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Jones, Leandro Roberto. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Quarleri, Jorge Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina

Traballo fin de grao (UDC.CIE). Bioloxía. Curso 2016/2017 [Resumen] La presencia de variantes asociadas a resistencia (VAR) en la región NS5A puede comprometer la eficacia de los antivirales de acción directa (AADs). La resistencia en el virus de la hepatitis C (VHC) está principalmente enfocada para pacientes con VHC y genotipos 1a (G1a) y 3 (G3) con otros factores de peor respuesta (e.j. cirrosis, exposición previa a tratamiento, ARN-VHC>800.000 UI/mL). En este estudio se evaluó, en una cohorte española de pacientes con VHC G1a y G3, la prevalencia de VAR basales en el dominio I de NS5A y su impacto en la optimización de las estrategias terapéuticas actuales. Las VAR se determinaron mediante secuenciación poblacional. Se consideraron como clínicamente relevantes las siguientes VAR: M28A/G/T, Q30D/E/H/G/K/L/R, L31M/V/F, H58D y Y93C/H/N/S para G1a y Y93H para G3. Un total de 232 pacientes fueron incluidos (166 G1a y 66 G3). La prevalencia de VAR a inhibidores de NS5A para pacientes con G1a y G3 fue baja (5,5%) o nula, respectivamente. Una alta proporción de pacientes tenían otros factores de peor respuesta asociados. En general, el número de pacientes que tenían VAR basales a inhibidores de NS5A en combinación con alguno de los otros factores fue bajo y la gran mayoría de los pacientes (G1a>94% y G3 100%) podrían recibir tratamientos estándar de 12 semanas sin ribavirina. El estudio de VAR en la región de NS5A en poblaciones específicas con infección por VHC (e.g. G1a & G3, cirrosis, exposición previa a tratamiento, ARN-VHC>800.000 UI/mL) puede ser útil para optimizar las estrategias terapéuticas basadas en inhibidores de NS5A evitando las toxicidades asociadas a la ribavirina y acortando la duración del tratamiento en la mayoría de los pacientes. [Resumo] A presenza de variantes asociadas a resistencia (VAR) na rexión NS5A pode comprometer a eficacia dos antivirais de acción directa (AADs). A resistencia no virus da hepatite C (VHC) está principalmente enfocada en pacientes con VHC e xenotipos 1a (G1a) e 3 (G3) con outros factores de peor resposta (e.g. cirrosis, exposición previa a tratamento, ARN-VHC>800.000 UI/mL). Neste estudo avaliouse, nunha cohorte española de doentes con VHC G1a e G3, a prevalencia de VAR basais no dominio I de NS5A e o seu impacto na optimización das estratexias terapéuticas actuais. As VAR determináronse mediante secuenciación poboacional. Consideráronse como clínicamente relevantes as seguintes VAR: M28A/G/T, Q30D/E/H/G/K/L/R, L31M/V/F, H58D e Y93C/H/N/S para G1a e Y93H para G3. Un total de 232 doentes foron incluidos (166 G1a e 66 G3). A prevalencia de VAR a inhibidores de NS5A para doentes con G1a e G3 foi baixa (5,5%) ou nula, respectivamente. Unha alta proporción de doentes tiñan outros factores de peor resposta asociados. En xeral, o número de doentes que tiñan VAR basais a inhibidores de NS5A en combinación con algún dos outros factores foi baixo e a gran maioría dos doentes (G1a>94% e G3 100%) poderían recibir tratamentos estándar de 12 semanas sen ribavirina. O test de VAR na rexión de NS5A en poboacións específicas con infección por VHC (e.g. G1a & G3, cirrosis, exposición previa a tratamento, ARN-VHC>800.000 UI/mL) pode ser útil para optimizar as estratexias terapéuticas baseadas en inhibidores de NS5A evitando as toxicidades asociadas á ribavirina e acurtando a duración do tratamento na maioría dos doentes. [Abstract] The presence of resistance-associated substitutions (RASs) at NS5A region might compromise the efficacy of Direct Acting Antiviral agents (DAAs). Hepatitis C virus (HCV) resistance at NS5A region is mainly focused on patients with HCV genotypes 1a (G1a) and 3 (G3) with other factors of poor treatment response (i.e. cirrhosis, prior treatment-exposure, or HCV-RNA>800,000 IU/mL). Herein, we evaluated in a Spanish cohort of patients with HCV G1a and G3 the prevalence of RASs at domain I NS5A using population-based sequencing and the impact of RASs on the optimization of current therapeutic strategies. RASs considered as clinically relevant were: M28A/G/T, Q30D/E/H/G/K/L/R, L31M/V/F, H58D and Y93C/H/N/S for G1a and Y93H for G3. A total of 232 patients were included (166 G1a and 66 G3). The overall prevalence of NS5A RASs for G1a and G3 patients was low (5.5%) or null, respectively. A high proportion of patients harbored other factors of poor response. Overall, the rates of patients harboring NS5A RASs in combination with any of the other factors were low and the vast majority of patients (G1a> 94% and G3 100%) could be treated with standard treatments of 12 weeks without ribavirin. Testing NS5A RASs in specific HCV-infected populations (i.e. G1a & G3, cirrhosis, prior treatment experienced, HCV-RNA>800.000 IU/mL) might be useful to optimize current NS5A-based therapies avoiding ribavirin-related toxicities and shortening treatment duration in the majority of patients.

Naturally occurring resistance-associated variants (RAVs) within the protease domain of hepatitis C virus (HCV) genotype (G) 1a separated into clades 1 and 2, and G1b were investigated in 59 HIV/HCV coinfected patients. RAVs were detected in 10/23 G1a/clade 1 and 1/19 G1b (p 0.0059). A similar frequency of RAVs was found when comparing G1a/clade 2 and G1b (p 0.1672). A cross-resistance to the macrocyclic compounds simeprevir and paritaprevir was detected in two G1a/clade 2 and 1 G1b sequences and none of G1a/clade 1 sequences. The simultaneous characterization of subtype and natural RAVs by population analysis of the NS3 domain by may add important information for anti-HCV treatment strategies including protease inhibitors.

The mechanisms related to the spontaneous clearance of hepatitis C virus (HCV) have been primarily studied in regions where the infection is endemic. Results of prior studies have been extrapolated to populations with low endemicity, such as Mexico. Herein, we determined the cytokine profiles in serum samples from Mexican patients who spontaneously cleared HCV and patients chronically infected with HCV genotype 1a. Chronic HCV-infected patients displayed increased interleukin (IL)-8 and regulated upon activation, normal T-cell expressed and secreted (CCL-5) secretion, whereas patients who spontaneously cleared HCV showed augmented levels of IL-1 alpha, tumour necrosis factor-alpha, transforming growth factor-beta, monocyte chemoattractant protein-2 (CCL-8), IL-13 and IL-15. Our study suggeststhat cytokine profiles may predict disease outcome during HCV infection.

Background. Hepatitis C virus (HCV) genotype 1 is the most prevalent worldwide. Subtype 1a, compared with 1b, shows lower response rates and higher propensity to select for drug resistance to NS3 and selected NS5A and nonnucleoside NS5B inhibitors. Two distinct clades of subtype 1a have been described.Methods. Using Bayesian methodology, we performed a time-scaled phylogeny reconstruction of clade separation and characterized the geographic distribution, phylodynamics, and association with natural resistance variants of NS3 sequences from 362 patients carrying subtype 1a HCV.Results. All sequences segregated in 2 clearly distinct clades. Clade I showed an earlier origin from the common ancestor compared with clade II. Clade I virus was more prevalent in non-European countries, represented mostly by United States, compared with European (75.7% vs 49.3%; P < .001). The prevalence of the natural NS3 variant Q80K, associated with resistance to the macrocyclic protease inhibitor simeprevir, was detected in 51.6% of clade I and 0% of clade II (P < .001); clade I showed a lower genetic barrier for Q80K, whereas no sign of selective pressure at any protease inhibitor resistance-associated codon was detected.Conclusions. Hepatitis C virus subtype 1a clades have a clearly different distribution in Europe and the United States, and the natural resistance mutation Q80K is exclusively associated with clade I.

The mechanisms related to the spontaneous clearance of hepatitis C virus (HCV) have been primarily studied in regions where the infection is endemic. Results of prior studies have been extrapolated to populations with low endemicity, such as Mexico. Herein, we determined the cytokine profiles in serum samples from Mexican patients who spontaneously cleared HCV and patients chronically infected with HCV genotype 1a. Chronic HCV-infected patients displayed increased interleukin (IL)-8 and regulated upon activation, normal T-cell expressed and secreted (CCL-5) secretion, whereas patients who spontaneously cleared HCV showed augmented levels of IL-1 alpha, tumour necrosis factor-alpha, transforming growth factor-beta, monocyte chemoattractant protein-2 (CCL-8), IL-13 and IL-15. Our study suggests that cytokine profiles may predict disease outcome during HCV infection.

The evolution of hepatitis C virus (HCV) quasispecies in patients with HIV-1 coinfection is not fully understood. The HCV-1a quasispecies heterogeneity was analyzed at inter and intra-host levels along 7.6 years in 21 coinfected patients that showed different virological and immunological responses to highly active antiretroviral therapy (HAART). Two to nine serial samples were subjected to direct and clonal sequence analyses of the envelope glycoprotein 2 (E2) gene. E2-based phylogenies, intra-host HCV evolution and evolutionary rates, as well as dynamics of the quasispecies heterogeneity parameters were evaluated. Bayesian coalescent phylogenies indicated complex evolutionary histories, revealing some viral lineages that persisted along the follow up and others that were detectable at a single or some sampling times, suggesting the occurrence of emergence-extinction cycles. HCV quasispecies underwent very rapid evolution in HAART-treated patients (~3.1 × 10(-2) sub/site/year) following the recovery of the host immunocompetence irrespectively of the virological response to HAART. Fil: Sede, Mariano Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: Jones, Leandro Roberto. Universidad Nacional de la Patagonia ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Moretti, Franco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: Laufer, Natalia Lorna. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: Quarleri, Jorge Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina

Parts of 5' non-coding (5' NC) and of E1 envelope regions of the hepatitis C virus (HCV) genome were amplified from sera of 26 Brazilian anti-HCV antibody-positive patients using the reverse transcription-polymerase chain reaction (RT-PCR). Fourteen samples were PCR positive with primers from the 5' NC region and 8 of them were also positive with primers from the E1 region. A genomic segment of 176 bp from the E1 region of 7 isolates was directly sequenced from PCR products. The sequences were compared with those of HCV strains isolated in other countries and the Brazilian isolates were classified by phylogenetic analysis into genotypes 1a and 1b. This could have a clinical importance since it has been shown that individuals infected with type 1 viruses are less likely to respond to treatment with interferon than individuals infected with types 2 and 3 viruses. Two quasispecies isolated from the same patient with an interval of 13 months differed by two base substitutions (1.1%). The sequence of another isolate presented a three-nucleotide deletion at codon 329.

New direct acting antivirals (DAAs) such as daclatasvir (DCV; BMS-790052), which target NS5A function with picomolar potency, are showing promise in clinical trials. The exact nature of how these compounds have an inhibitory effect on HCV is unknown; however, major resistance mutations appear in the N-terminal region of NS5A that include the amphipathic helix and domain 1. The dimeric symmetry of these compounds suggests that they act on a dimer of NS5A, which is also consistent with the presence of dimers in crystals of NS5A domain 1 from genotype 1b. Genotype 1a HCV is less potently affected by these compounds and resistance mutations have a greater effect than in the 1b genotypes. We have obtained crystals of domain 1 of the important 1a NS5A homologue and intriguingly, our X-ray crystal structure reveals two new dimeric forms of this domain. Furthermore, the high solvent content (75%) makes it ideal for ligand-soaking. Daclatasvir (DCV) shows twofold symmetry suggesting NS5A dimers may be of physiological importance and serve as potential binding sites for DCV. These dimers also allow for new conformations of a NS5A expansive network which could explain its operation on the membranous web. Additionally, sulfates bound in the crystal structure may provide evidence for the previously proposed RNA binding groove, or explain regulation of NS5A domain 2 and 3 function and phosphorylation, by domain 1.

Background and aim. Resistance-associated variants (RAVs) on the NS3 region of the hepatitis C virus (HCV) may be relevant for antiviral therapy, but data in human immunodeficiency virus (HIV) coinfected patients are scarce. We assessed frequencies of NS3 RAVs in patients infected with HCV genotype 1a with or without HIV coinfection. MATERIAL AND METHODS: HCV NS3 amino acids 1-181 were sequenced by the Sanger method and analyzed for RAVs. RAVs and their distribution between HCV genotype 1a clade I and II viruses were compared between HIV-infected versus HIV-uninfected patients. RESULTS: 148 samples were available (n = 68 HIV and n = 80 non-HIV). Relative frequency of clade I and clade II was significantly different between HIV (85% and 15%) and non-HIV groups (49% and 51%). Overall, HIV infected patients exhibited significantly lower prevalence of RAVs than HIV-uninfected patients (62% vs. 79%, p = 0.03). However, Q80K prevalence was significantly higher in HIV-infected subjects (50% vs. 24%, p = 0.001), whereas prevalence of S122D/G/N/S (2% vs. 16%, p = 0.002) and N174G/N/S (10% vs. 55%, p < 0.0001) polymorphisms were significantly lower. Q80K was found exclusively in clade I viruses. S122 (3% vs. 22%, p=0.001) and N174 (13% vs. 75%, p

Chronic coinfection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) is among the greatest challenges facing public health worldwide. In this population, the response to hepatitis C therapy by treatment with pegylated interferon plus ribavirin (PEG-IFN+RBV) is lower than in HCV-monoinfected patients, particularly in those infected by HCV genotype 1. A PKR/eIF-2α phosphorylation homology domain (PePHD) within the E2 protein has been found to interact with PKR and inhibit PKR in vitro, suggesting a possible mechanism for HCV to evade the antiviral effects of IFN. The aim of this work was to analyze the amino acid conservation in the HCV-E2-PePHD and quasispecies diversity among HCV-HIV-coinfected patients exhibiting sustained virological response, non-response, or partial response with viral relapse to PEG-IFN+RBV by ultra-deep pyrosequencing. For this purpose, HCV-E2-PePHD PCR products were generated and sequenced directly for four patients with a sustained response, seven patients with no virological response, and four patients with viral relapse before and after treatment with PEG-IFN+RBV. HCV-E2-PePHD amino acid sequences were obtained for isolates from serum collected before and during treatment (24 h, 4 weeks, and 12 weeks). Quasispecies analysis of the HCV-E2-PePHD and flanking genomic regions was performed using 454/Roche pyrosequencing, analyzing 39,364 sequence reads in total. The HCV-E2-PePHD sequence at the amino acid and nucleotide level was highly conserved among HCV genotype 1 strains, irrespective of the PEG-IFN+RBV response. This high degree of amino acid conservation and sporadic mutations in the HCV-E2-PePHD domain do not appear to be associated with treatment outcome. The HCV-E2-PePHD sequence before or during treatment cannot be used to predict reliably the outcome of treatment in patients coinfected with HCV genotype 1 and HIV. © 2012 Springer-Verlag. Fil: Bolcic, Federico Martin. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Centro Nacional de Referencia para el Sida; Argentina Fil: Sede, Mariano Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Centro Nacional de Referencia para el Sida; Argentina Fil: Moretti, Franco. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Westergaard, G.. Instituto de Agrobiotecnología Rosario; Argentina Fil: Vazquez, Martin Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Agrobiotecnología Rosario; Argentina Fil: Laufer, Natalia Lorna. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Centro Nacional de Referencia para el Sida; Argentina Fil: Quarleri, Jorge Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Centro Nacional de Referencia para el Sida; Argentina

Background The impact of viral subtype on the rate of sustained virological response (SVR) to antiviral therapy in patients chronically infected with hepatitis C genotype 1 subtype 1a and 1b has not been extensively investigated. The aim of this study is to determine whether the HCV genotype 1 subtypes 1a and 1b respond differently to treatment with PEGylated interferon (PEG-IFN) plus ribavirin. Methods For 48 weeks, 388 “naïve”genotype 1 patients were treated weekly with PEG-IFN α-2a or PEG-INF α-2b combined with daily ribavirin (1000–1200 mg/day). The numbers of patients in whom HCV-RNA was undetectable were compared after 4 (rapid virological response, RVR), 12 (early virological response, EVR), and 48 (end treatment virological response, ETR) weeks of treatment as well as 24 weeks after the last treatment (sustained virological response, SVR). Results The rate of SVR was higher in subtype 1a patients than subtype 1b patients (55% vs. 43%; p 10 IU/ml (OR: 3.2; 95% CI: 2.7 to 6.9) and fibrosis score < S3 (OR: 3.8; 95% CI:3.2 to 7.4), were all independent predictors of SVR. Conclusion Dual antiviral therapy is more effective against HCV subtype 1a than against subtype 1b and this difference is independent of other factors that may favour viral clearance. Trial registration ClinicalTrials.gov Identifier: NCT01342003